Trans-acting factors that mediate B cell specific transcription of immunoglobulin (Ig) genes have been postulated based on an analysis of the expression of exogenously introduced Ig gene recombinants in lymphoid and non-lymphoid cells. Two B cell-specific, cis-acting transcriptional regulatory elements have been identified. One element is located in the intron between the variable and constant regions of both heavy and kappa light chain genes and acts as a transcriptional enhancer. The second element is found upstream of both heavy chain and kappa light chain gene promoters. This element directs lymphoid-specific transcription even in the presence of viral enhancers.
Mouse and human light chain promoters contain the octamer sequence ATTTGCAT approximately 70 base pairs upstream from the site of initiation. Heavy chain gene promoters contain the identical sequence in inverted orientation, ATGCAAAT, at the same position. This element appears to be required for the efficient utilization of Ig promoters in B cells. The high degree of sequence and positional conservation of this element as well as its apparent functional requirement suggests its interaction with a sequence-specific transcription factor but no such factor has been identified.
This invention pertains to human lymphoid-cell nuclear factors which bind to gene elements associated with regulation of the transcription of Ig genes and to methods for identification and for isolation of such factors. The factors are involved in the regulation of transcription of Ig genes. The invention also pertains to the nucleic acid encoding the regulatory factors, to methods of cloning factor-encoding genes and to methods of altering transcription of Ig genes in lymphoid cells or lymphoid derived cells, such as hybridoma cells, by transfecting or infecting cells with nucleic acid encoding the factors.
Four different factors which bind to transciptional regulatory DNA elements of Ig genes were identified and isolated in nuclear extracts of lymphoid cells. Two of the factors, IgNF-A and E, are constitutive; two IgNF-B and xcexa-3 (hereinafter NF-xcexaB) are lymphoid cell specific. Each factor is described below.
IgNF-A (NF-A1)
IgNF-A binds to DNA sequences in the upstream regions of both the murine heavy and kappa light chain gene promoters and also to the murine heavy chain gene enhancer. The binding is sequence specific and is probably mediated by a highly conserved sequence motif, ATTTGCAT, present in all three transcriptional elements. A factor with binding specificity similar to IgNF-A is also present in human HeLa cells indicating that IgNF-A may not be tissue specific.
E Factors
The E factors are expressed in all cell types and bind to the light and heavy chain enhancers.
IgNF-B (NF-A2)
IgNF-B exhibits the same sequence-specificity as IgNF-A; it binds to upstream regions of murine heavy and kappa light chain gene promoters and to murine heavy chain gene enhancer. This factor, however, is lymphoid specific; it is restricted to B and T cells.
NF-xcexaB (Previously Kappa-3)
NF-xcexaB binds exclusively to the kappa light chain gene enhancer (the sequence TGGGGATTCCCA). Initial work provided evidence that NF-kB is specific to B-lymphocytes (B-cells) and also to be B-cell stage specific. NF-kB was originally defected because it stimulates transcription of genes encoding kappa immunoglobulins in B lymphocytes. As described herein, it has subsequently been shown that transcription factor NF-kB, previously thought to be limited in its cellular distribution, is, in fact, present and inducible in many, if not all, cell types and that it acts as an intracellular messenger capable of playing a broad role in gene regulation as a mediator of inducible signal transduction. It has now been demonstrated that NF-kB has a central role in regulation of intercellular signals in many cell types. For example, NF-kB has not been shown to positively regulate the human xcex2-interferon (xcex2-IFN) gene in many, if not all, cell types. As described below, it is now clear not only that NF-kB is not tissue specific in nature, but also that in the wide number of types of cells in which it is present, it serves the important function of acting as an intracellular transducer of external influences. NF-kB has been shown to interact with a virus inducible element, called PRDII, in the xcex2-IFN gene and to be highly induced by virus infection or treatment of cells with double-stranded RNA. In addition, NF-kB controls expression of the human immunodeficiency virus (HIV).
As further described, it has been shown that a precursor of NF-KB is present in a variety of cells, that the NF-KB precursor in cytosolic fractions is inhibited in its DNA binding activity and that inhibition can be removed by appropriate stimulation, which also results in translocation of NF-KB to the nucleus. A protein inhibitor of NF-KB, designated IkB, has been shown to be present in the cytosol and to convert NF-KB into an inactive form in a reversible, saturable and specific reaction. Release of active NF-kB from the IkB-NF-kB complex has been shown to result from stimulation of cells by a variety of agents, such as bacterial lipopolysaccharide, extracellular polypeptides and chemical agents, such as phorbel esters, which stimulate intracellular phosphokinases. IkB and NF-KB appear to be present in a stoichiometric complex and dissociation of the two complex components results in two events: activation (appearance of NF-KB binding activity) and translocation of NF-KB to the nucleus.
Identification and Isolation of the Transcriptional Regulatory Factors
The transcription regulatory factors of the present invention were identified and isolated by means of a modified DNA binding assay. The assay has general applicability for analysis of protein DNA interactions in eukaryotic cells. In performing the assay, DNA probes embodying the relevant DNA elements or segments thereof are incubated with cellular nuclear extracts. The incubation is performed under conditions which allows the formation of protein-DNA complexes. Protein-DNA complexes are resolved from uncomplexed DNA by electrophoresis through polyacrylamide gels in low ionic strength buffers. In order to minimize binding of protein in a sequence nonspecific fashion, a competitor DNA species can be added to the incubation mixture of the extract and DNA probe. In the present work with eukaryotic cells the addition of alternating copolymer duplex poly(dI-dC)-poly(dI-dC) as a competitor DNA species provided for an enhancement of sensitivity in the detection of specific protein-DNA complexes and facilitated detection of the regulatory factors described herein.
This invention pertains to the transcriptional regulatory factors, the genes encoding the four factors associated with transcriptional regulation, reagents (e.g., oligonucleotide probes, antibodies) which include or are reactive with the genes or the encoded factors and uses for the genes, factors and reagents. It further relates to NF-KB inhibitors, including isolated IkB, the gene encoding IkB and agents or drugs which enhance or block the activity of NF-KB or of the NF-KB inhibitor (e.g., IkB).
The invention also pertains to a method of cloning DNA encoding the transcriptional regulatory factors or other related transcriptional regulatory factors. The method involves screening for expression of the part of the binding protein with binding-site DNA probes. Identification and cloning of the genes can also be accomplished by conventional techniques. For example, the desired factor can be purified from crude cellular nuclear extracts. A portion of the protein can then be sequenced and with the sequence information, oligonucleotide probes can be constructed and used to identify the gene coding the factor in a cDNA library. Alternatively, the polymerase chain reaction (PCR) can be used to identify genes encoding transcriptional regulatory factors.
The present invention further relates to a method of inducing expression of a gene in a cell. In the method, a gene of interest (i.e., one to be expressed) is linked to the enhancer sequence containing the NF-KB binding site in such a manner that expression of the gene of interest is under the influence of the enhancer sequence. The resulting construct includes the kappa enhancer or a kappa enhancer portion containing at least the NF-KB binding site, the gene of interest, and a promoter appropriate for the gene of interest. Cells are transfected with the construct and, at an appropriate time, exposed to an appropriate inducer of NF-KB, resulting in induction of NF-KB and expression of the gene of interest.
The subject invention further relates to methods of regulating (inducing or preventing) activation of NF-KB, controlling expression of the immunoglobulin kappa light chain gene and of other genes whose expression is controlled by NF-KB (e.g., HIV).
As a result of this finding, it is now possible to alter or modify the activity of NF-xcexaB as an intracellular messenger and, as a result, to alter or modify the effect of a variety of external influences, referred to as inducing substances, whose messages are transduced within cells through NF-xcexaB activity. Alteration or modification, whether to enhance or reduce NF-xcexaB activity or to change its binding activity (e.g., affinity, specificity), is referred to herein as regulation of NF-xcexaB activity. The present invention relates to a method of regulating or influencing transduction, by NF-xcexaB, of extracellular signals into specific patterns of gene expression and, thus, of regulating NF-xcexaB-mediated gene expression in the cells and systems in which it occurs.
In particular, the present invention relates to a method of regulating (enhancing or diminishing) the activity of NF-xcexaB in cells in which it is present and capable of acting as an intracellular messenger, as well as to substances or composition useful in such a method. Such methods and compositions are designed to make use of the role of NF-xcexaB as a mediator in the expression of genes in a variety of cell types. The expression of a gene having a NF-xcexaB binding recognition sequence can be regulated, either positively or negatively, to provide for increased or decreased production of the protein whose expression is mediated by NF-xcexaB. NF-xcexaB-mediated gene expression can also be selectively regulated by altering the binding domain of NF-xcexaB in such a manner that binding specificity and/or affinity are modified. In addition, genes which do not normally possess NF-xcexaB binding recognition sequences can be placed under the control of NF-xcexaB by inserting an NF-xcexaB binding site in an appropriate position, to produce a construct which is then regulated by NF-xcexaB. As a result of the present invention, cellular interactions between NF-xcexaB and a gene or genes whose expression is mediated by NF-xcexaB activity and which have, for example, medical implications (e.g., NF-xcexaB/cytokine interactions; NF-xcexaB/HTLV-I tax gene product interactions) can be altered or modified.
Genes encoding the regulatory factors can be used to alter cellular transcription. For example, positive acting lymphoid specific factors involved in Ig gene transcription can be inserted into Ig-producing cells in multiple copies to enhance Ig production. Genes encoding tissue specific factors can be used in conjunction with genes encoding constitutive factors, where such combinations are determined necessary or desirable. Modified genes, created by, for example, mutagenesis techniques, may also be used. Further, the sequence-specific DNA binding domain of the factors can be used to direct a hybrid or altered protein to the specific binding site.
DNA sequences complementary to regions of the factor-encoding genes can be used as DNA probes to determine the presence of DNA encoding the factors for diagnostic purposes and to help identify other genes encoding transcriptional regulatory factors. Antibodies can be raised against the factors and used as probes for factor expression. In addition, the cloned genes permit development of assays to screen for agonists or antagonists of gene expression and/or of the factors themselves. Further, because the binding site for NF-kB in the kappa gene is clearly defined, an assay for blockers or inhibitors of binding is available, as is an assay to determinte whether active NF-kB is present.